POOR EXPRESSION OF MDR1 TRANSGENE IN HELA-CELLS BY BICISTRONIC MOLONEY MURINE LEUKEMIA VIRUS-BASED VECTOR

Cotransfer of a therapeutic gene together with the human MDR1 gene pro vides an opportunity to increase the number of transduced marrow cells , expressing the therapeutic gene, by in vivo selection for MDR1. We h ave used an Lg-MDR1-IRES-neo (LgMIN) retroviral vector, containing MDR 1 and neo genes, separated by the EMCV IRES. Human HeLa or canine CTAC cells, transduced with GALV env pseudotyped LgMIN at an MOI of less t han 0.01 to ensure 1 proviral copy/genome, were selected with either G 418 for neo expression or colchicine for MDR1 expression. The titer de termined on HeLa cells with G418 selection was eightfold higher than t hat with colchicine selection. In contrast, the same viral supernatant exhibited only a 1.4-fold difference between neo- and MDR1-based vira l titer values for CTAC cells. The transduced HeLa cells, with one int act proviral copy per genome, exhibited a 55-fold higher resistance to G418 but only a 4-fold higher resistance to colchicine and a 2-fold h igher resistance to Taxol compared with nontransduced cells. About 23% of the transduced cell population did not express vector-derived P-gl ycoprotein (P-gp) as detected by anti-human P-gp MAb MRK-16. This coul d explain the difference in viral titers obtained on CTAC cells but no t that obtained on HeLa cells. The vector-mediated increase in express ion of P-gp was about 20-fold higher in CTAC cells as compared with He La cells. These results indicated suppression of expression of vector- derived MDR1 in HeLa cells, in contrast with CTAC cells. To investigat e further the possible reasons for this difference, genomic DNA was is olated from the G418-resistant individual colonies of infected cells a nd analyzed by PCR for full-length proviral MDR1. For transduced CTAC and HeLa cells, selected at a G418 concentration of 1 mg/ml, PCR detec ted aberrant forms of MDR1 in 17 to 25% of colonies tested. The aberra nt forms consisted of MDR1 genes with 2- and 0.7-kb deletions. DNA seq uencing across the 2-kb and the 0.7-kb deletion junction suggests cryp tic splicing in the producer cell line as the origin of these deletion s. The 2-kb deletion corresponds to MDR1 mRNA cryptic splicing via don or (codon 113) and acceptor (codon 773). The 0.7-kb deletion correspon ds to splicing via the same donor and a different acceptor (codon 344) . When transduced HeLa cells were selected at a higher concentration o f G418 (3 mg/ml), the aberrant forms were detected at an increased fre quency of about 50% of colonies tested. These results indicate that ve ctor-derived MDR1 is a poor selective marker in HeLa cells but not in CTAC cells and that deletions, which inactivated the MDR1 gene in a bi cistronic Mo-MuLV vector, may provide an advantage for expression of t he second transgene in HeLa cells.